Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design

Pryor, John M.; Потапов, В. А.; Kucera, Rebecca; Bilotti, Katharina; Cantor, Eric J.; Lohman, Gregory J. S.

doi:10.1371/journal.pone.0238592

Cited by 52 publications

(104 citation statements)

References 35 publications

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“…sgRNAs TUs are prepared in shuttle vectors by cloning of hybridized oligonucleotides via a Bpi I/ Bbs I GoldenGate reaction (Figure 1b). In shuttle vectors containing an At U6‐26 promoter fragment, we exchanged the sgRNA scaffold (Dang et al , 2015) and modified an overhang that was identified as sub‐optimal using web tools (http://www.tools.neb.com; Pryor et al , 2020). We also generated additional shuttle vectors containing either U3 or U6 promoter fragments from S. lycopersicum ( Sl U6/U3).…”

Section: Resultsmentioning

confidence: 99%

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

et al. 2021

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SUMMARY Genome editing by RNA‐guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron‐optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene‐free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter‐selection markers for N. benthamiana, most importantly Sl‐FAST2, comparable to the well‐established Arabidopsis seed fluorescence marker, and FCY‐UPP, based on the production of toxic 5‐fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY‐UPP for selection of non‐transgenic T1, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher‐order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.

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Section: Resultsmentioning

confidence: 99%

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

et al. 2021

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“…S1 ). Most published protocols for Golden Gate assembly are designed for the assembly of fragments from circular vectors, but not from linear dsDNA like in our case 46 . We therefore optimised each stage of the concatenation process to ensure sufficient yield of the fully assembled 5 × amplicon.…”

Section: Resultsmentioning

confidence: 99%

PacBio sequencing output increased through uniform and directional fivefold concatenation

Kanwar

Blanco

Chen

et al. 2021

Sci Rep

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Advances in sequencing technology have allowed researchers to sequence DNA with greater ease and at decreasing costs. Main developments have focused on either sequencing many short sequences or fewer large sequences. Methods for sequencing mid-sized sequences of 600–5,000 bp are currently less efficient. For example, the PacBio Sequel I system yields ~ 100,000–300,000 reads with an accuracy per base pair of 90–99%. We sought to sequence several DNA populations of ~ 870 bp in length with a sequencing accuracy of 99% and to the greatest depth possible. We optimised a simple, robust method to concatenate genes of ~ 870 bp five times and then sequenced the resulting DNA of ~ 5,000 bp by PacBioSMRT long-read sequencing. Our method improved upon previously published concatenation attempts, leading to a greater sequencing depth, high-quality reads and limited sample preparation at little expense. We applied this efficient concatenation protocol to sequence nine DNA populations from a protein engineering study. The improved method is accompanied by a simple and user-friendly analysis pipeline, DeCatCounter, to sequence medium-length sequences efficiently at one-fifth of the cost.

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“…sgRNAs TUs are prepared in shuttle vectors by cloning of hybridized oligonucleotides via a Bpi I/ Bbs I GoldenGate reaction (Figure 1b). In shuttle vectors containing an At U6-26 promoter fragment, we exchanged the sgRNA scaffold (Dang et al , 2015) and modified an overhang that was identified as sub-optimal using web tools (www.tools.neb.com; Pryor et al , 2020). We also generated additional shuttle vectors containing either U3 or U6 promoter fragments from tomato ( S. lycopersicum; Sl U6/U3).…”

Section: Resultsmentioning

confidence: 99%

Highly efficient multiplex editing: One-shot generation of 8xNicotiana benthamianaand 12x Arabidopsis mutants

Stuttmann

Barthel

Martin

et al. 2020

Preprint

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SummaryGenome editing by RNA-guided nucleases in model species is still hampered by low efficiencies, and isolation of transgene-free individuals often requires tedious PCR screening. Here, we present a toolkit that mitigates these drawbacks for Nicotiana benthamiana and Arabidopsis thaliana. The toolkit is based on an intron-optimized SpCas9-coding gene (zCas9i), which conveys dramatically enhanced editing efficiencies. The zCas9i gene is combined with remaining components of the genome editing system in recipient vectors, which lack only the user-defined guide RNA transcriptional units. Up to 32 guide RNA transcriptional units can be introduced to these recipients by a simple and PCR-free cloning strategy, with the choice of three different RNA polymerase III promoters for guide RNA expression. We developed new markers to aid transgene counter-selection in N. benthamiana, and demonstrate their efficacy for isolation of several genome-edited N. benthamiana lines. In Arabidopsis, we explore the limits of multiplexing by simultaneously targeting 12 genes by 24 sgRNAs. Perhaps surprisingly, the limiting factor in such higher order multiplexing applications is Cas9 availability, rather than recombination or silencing of repetitive sgRNA TU arrays. Through a combination of phenotypic screening and pooled amplicon sequencing, we identify transgene-free duodecuple mutant Arabidopsis plants directly in the T2 generation. This demonstrates high efficiency of the zCas9i gene, and reveals new perspectives for multiplexing to target gene families and to generate higher order mutants.

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Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design

Cited by 52 publications

References 35 publications

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

PacBio sequencing output increased through uniform and directional fivefold concatenation

Highly efficient multiplex editing: One-shot generation of 8xNicotiana benthamianaand 12x Arabidopsis mutants

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